Levetiracetam (LEV) ((−)-(S)-α-ethyl-2-oxo-1-pyrrolidine acetamide, Keppra®) was approved by FDA in 2006 as the first-in-class anti-epileptic drug (AED). The compound is marketed under the trade name Keppra® by UCB Pharmaceuticals, Inc.
Levetiracetam is indicated as adjunctive therapy in the treatment of partial onset seizures in adults and children 4 years of age and older, myoclonic seizures in adults and adolescents 12 years of age and older, and primary generalized tonic-clonic seizures in adults and children 6 years of age and older. LEV acts by modulating synaptic neurotransmitter release by binding to the synaptic vesicle protein SV2A in the brain (1). The nominal therapeutic range for LEV is 6-20 ug/mL (2). Therapeutic drug monitoring (TDM) of LEV is important in order to establish an individual's optimum LEV level (3) and to detect non-compliance.
An optimal trough serum/plasma concentration range for LEV is 10-60 mg/L (4-6) LEV is predominantly eliminated via the kidney with approximately 64% of a given dose excreted unchanged in urine. The drug undergoes minimal hepatic metabolism, but hydrolysis of the acetamide function by a cytosolic amidase occurs to produce a carboxylic acid metabolite, 2-pyrrolidone-N-butyric acid. The acid metabolite is excreted in urine and accounts for approximately 27% of the administered dose. Oxidation of the 3 and 4 positions of the 2-oxopyrrolidine ring also occurs by hepatic metabolism to form minor metabolites that account for about 3% of the dose. In addition, LEV and 2-pyrrolidone-N-butyric acid may be oxidized at the 5 position of the 2-oxopyrrolidine ring and then hydrolyzed, resulting in opening of the ring. There is pronounced inter-individual variability in LEV pharmacokinetics. Therefore, therapeutic monitoring of serum/plasma concentration of LEV is recommended.
To date, liquid or gas chromatographic techniques have been employed to measure circulating levels of LEV. However, such methods are impractical for regular TDM due to factors including long sample preparation time, long assay time, high cost, low throughput and labor-intensive procedures. Thus, a fast economical analytical method for measurement of plasma levels of LEV is needed for effective TDM.
Immunoassays are generally regarded as fast, inexpensive and sensitive means for quantifying levels of an antigen present in a sample. Immunoassay techniques have been developed to detect various drugs in biological samples and are well suited for such commercial analytical applications. Accordingly, immunoassays can be used to quickly determine the amount of a drug and/or drug metabolite in a patient's blood, serum, urine, saliva or other body fluids. Examples of immunoassays can include, but not limited to, homogeneous microparticle immunoassay (e.g., immunoturbidimetric) or quantitative microsphere systems (“QMS®”), fluorescence polarization immunoassay (“FPIA”), cloned enzyme donor immunoassay (“CEDIA”), chemiluminescent microparticle immunoassay (“CMIA”), and the like. It would be advantageous to have immunoassays configured to detect LEV in a patient's blood, serum, plasma, and/or other biological fluids or samples. Additionally, it would be advantageous to have LEV derivatives for use in such immunoassays, and/or LEV-based immunogens for use in producing anti-levetiracetam antibodies.
Because at least one antibody specific to the target antigen typically forms the basis of a useful immunoassay, TDM presents a challenge in that most therapeutic drugs are not, by nature, immunogenic and, therefore, do not elicit an immune response when injected into animals commonly used for antibody production such as mice, rabbits, goats, horses and other mammals. Also, active forms of the target drug may be detrimental to the recipient animal, even in small doses. Therefore, a derivative of the therapeutic drug must be made to serve as an immunogen. However, the antibodies produced in response to the immunogen must be able to cross-react with the drug expected to be present in patient samples and, even more preferably, the antibodies should have little, if any, measurable cross-reactivity with inactive metabolic derivatives of the drug that may also be present in the samples.
Immunoassays typically employ other types of derivatives in addition to the immunogen needed for antibody production. By way of non-limiting example, labeled derivatives of a target antigen may be used to compete with the target antigen for binding sites on an antibody capable of recognizing both the target antigen and the labeled derivative. Competitive immunoassays are well known in the current art. Other derivatives may serve as controls or as standards for calibrating the immunoassay.
Levetiracetam, its metabolites, and structurally similar drugs are shown below:

Kenda et al., in attempting to identify derivatives with a higher binding affinity for levetiracetam binding sites than LEV, describe derivatives of LEV including 5-amino-2-(2-oxopyrrolidin-1-yl)pentanoic acid amide (referred to as “analog 56” by Kenda et al.) produced via solid phase synthesis by reductive amination of aldehydic esters. As described, the reaction used 100 grams of rink amide resin and 66 mg of 4-oxobutyric acid 4-methoxybenzyl ester to yield only 2 mg of analog 56 with a purity of 65% as measured by HPLC for a reactive yield of about 0.1%.

While analog 56 may have promise as a derivative of LEV for use in immunoassays, in order to induce antibody production in a number of animals and monitor antibody production, binding properties and other characteristics, amounts significantly in excess of 2 mg of the immunogenic derivative are required. Likewise, commercialization of an immunoassay comprising a derivative used as a control or standard, or a labeled derivative to compete with target antigen, requires sufficient quantities of the derivative(s) to satisfy commercial demand. So, while the solid phase synthesis method taught by Kenda et al. may be suitable for drug discovery purposes, it has no practical value for producing derivatives for the purpose of developing an immunoassay for LEV.